This proposal sets forth a precise and rapid methodology for identifying the interaction sites between monoclonal antibodies and proteins. The basis of the system is a high-throughput mass spectrometry-based analysis of epitope determinants to identify the interacting protein sequences using immunoaffinity capture combined with enzymatic digestion of the target protein;either pre- or post-capture. This combination will be used to rapidly determine the approximate location of each binding epitope. The precise location of the binding epitope will then be determined by immunoaffinity capture and mass spectrometric readout of sets of synthetic ladder peptides that span the approximate epitope established from the initial analysis. The project will develop reproducible methods to demonstrate epitope mapping for monoclonal antibodies of interest to NCI tested by multiple sampling and quantifiable results. Mapped epitopes will be functionally characterized by amino acid substitutions in the epitope sequence using high-throughput spotted array surface plasmon resonance imaging and SPR-Biomolecular Interaction Analysis to identify the key amino acids that are critically involved in binding the antibody to the antigenic epitope. Thus, this approach will provide an efficient and cost effective platform from which to accomplish high-throughput epitope mapping of monoclonal antibodies for use in diagnosis of cancer.